Background: Extrapulmonary tuberculosis (TB) presents significant diagnostic challenges, particularly in the context of multidrug-resistant (MDR) strains. This study assessed the utility of the WHO-recommended rapid molecular assays, originally validated for pulmonary TB, in diagnosing extrapulmonary TB and detecting the MDR Mycobacterium tuberculosis complex (MTBC). Materials and Methods: A total of 6274 clinical samples, including 4891 pulmonary and 1383 extrapulmonary samples, were analyzed between 2019 and 2022 using the BD MAX™ MDR-TB assay (BD MAX), the Xpert^® MTB/RIF assay (Xpert MTB/RIF), the Xpert^® MTB/XDR assay (Xpert MTB/XDR), FluoroType MTB, and phenotypic drug susceptibility testing (DST). Results: MTBC was detected in 426 samples using BD MAX (376 pulmonary and 50 extrapulmonary), of which 277 were culture-confirmed. Phenotypic testing confirmed 299 positive cultures on Löwenstein–Jensen (LJ) medium and 347 in BD BACTEC™ MGIT™ (BACTEC MGIT) mycobacterial growth indicator tube (BBL) liquid culture. BD MAX showed high sensitivity and specificity for extrapulmonary TB detection (93.1% and 98.4%, respectively). Resistance to isoniazid or rifampicin was identified in 11% of MTBC-positive cases, whereas 3.69% were confirmed as MDR-TB. The molecular assays effectively detected resistance-associated mutations (katG, inhA, and rpoB), with high concordance to phenotypic tests (DST) (κ = 0.69–0.89). Conclusions: This study demonstrates that molecular assays, although validated for pulmonary TB, are also reliable for extrapulmonary TB detection and drug resistance profiling. Their rapid turnaround and robust accuracy support broader implementation in routine diagnostics, especially for challenging extrapulmonary specimens where early detection is critical for targeted therapy.

One of the most relevant and pathogenic groups among the rapidly growing mycobacteria (RGM) is Mycobacterium abscessus complex (MABC) that includes three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. The aim of this study was the analysis of prevalence of MABC among other non-tuberculous mycobacteria isolated from patients in the Malopolska Region of Poland, between 2018 and 2021, as well as determination of their subspecies and molecular mechanisms of resistance to macrolides and aminoglycosides. The incidence of MABC was 5,4% (12/223). Eight strains were classified as M. abscessus subsp. abscessus, three as M. abscessus subsp. massiliense and one M. abscessus subsp. bolletii. Molecular analysis showed resistance to macrolides for eight strains of M. abscessus subsp. abscessus associated with erm(41)T28 gene mutations. One strain of M. abscessus subsp. abscessus showed resistance to macrolides (two mutations simultaneously: in erm(41)T28 and rrl genes) and aminoglycosides (point mutation in rrs gene). One strain of M. abscessus subs. bolletii was resistant to macrolides (erm(41)T28 mutation), whereas presented no mutations for aminoglycosides. M. abscessus subsp. massiliense reveal no mutations. High clarithromycin resistance of M. abscessus, determines the urgent need for susceptibility-based treatment. Molecular determination of resistance mechanisms to aminoglycosides and macrolides enables fast and accurate targeted treatment implementation.

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Background/Objectives: Certain yeast species are recognized as significant opportunistic pathogens, capable of causing severe systemic infections, particularly in immunocompromised individuals or those with disrupted physiological barriers. The rising incidence of invasive candidiasis associated with vascular infections poses a significant clinical challenge due to the high mortality rates and the limited efficacy of conventional antifungal therapies. The formation of resilient biofilms on vascular catheters by species such as Candida albicans and Nakaseomyces glabratus further complicates treatment, often leading to persistent fungemia and necessitating device removal. With the emergence of multidrug-resistant (MDR) strains, there is a critical need for new therapeutic agents like rezafungin—a novel, long-acting echinocandin with potential enhanced antibiofilm activity. Methods: This study tested susceptibility to antimycotics available in Poland (fluconazole, voriconazole, posaconazole, amphotericin B, anidulafungin, caspofungin, and micafungin) using the commercial Micronaut-AM test (Bruker, Bremen, Germany). Susceptibility to rezafungin (Angene Chemical, Great Britain) was determined using the microdilution method in RPMI medium, recommended by European Committee on Antimicrobial Susceptibility Testing (EUCAST), with amphotericin B as a control compound. We evaluated the biofilm-forming capacity and the in vitro activity of rezafungin against 42 clinical isolates of Candida albicans and Nakaseomyces glabratus recovered from positive blood cultures. Results: The obtained minimum inhibitory concentration (MIC) values suggest rezafungin activity against all the tested isolates, with different susceptibility to echinocandins and other antifungal drugs (azoles, amphotericin B) currently registered and used in Poland. The MIC readings for rezafungin were in the range of 0.008–0.5, with MIC₅₀ = 0.016 and MIC₉₀ = 0.25. The isolates were categorized as low, moderate, or strong biofilm producers according to established Stepanović criteria (cut-off values OD₆₃₀ < 0.019, 0.19–0.38, >0.38, respectively). Furthermore, the higher minimum biofilm eradication concentrations (MBECs) compared to the minimum inhibitory concentrations (MICs) of planktonic cells confirm the reduced activity of rezafungin against biofilms. Conclusions: Critically, the high antibiofilm efficacy at clinically achievable concentrations suggests that rezafungin shows promise as a potential therapeutic option for catheter-related candidemia, though further clinical studies are needed. Furthermore, the high susceptibility of N. glabratus isolates—a species frequently associated with azole resistance—suggests rezafungin may be a valuable addition to the existing antifungal arsenal of multidrug-resistant (MDR) fungal infections in hospital settings. Future research should focus on in vivo models to confirm if these in vitro results translate into accelerated clearance of vascular biofilms.

Mycobacterium abscessus complex (MABc) poses a major therapeutic challenge due to its intrinsic multidrug resistance and ability to form biofilms. This study evaluated the in vitro activity of three antimycobacterial agents—bedaquiline, delamanid, and clofazimine—on 20 clinical MABc isolates, including M. abscessus subsp. abscessus, massiliense, and bolletii, with a focus on biofilm-forming phenotypes. Biofilm analysis showed that the rough colony morphotypes were mostly weak biofilm formers, while the smooth and mixed morphotypes were predominantly moderate or strong biofilm formers. A statistically significant association was observed between the mixed colony morphology and strong biofilm formation (p = 0.032). Importantly, bedaquiline exhibited potent and consistent activity across all isolates, regardless of the biofilm-forming ability, with MIC values ranging from 0.125 to 1 µg/mL. In contrast, delamanid and clofazimine showed limited efficacy, with MIC values exceeding 16 µg/mL and 8 µg/mL, respectively. These findings strongly support the role of bedaquiline as a promising core agent for future combination therapies targeting drug-resistant MABc infections, including biofilm-associated infections. Our results, among the first from Poland, highlight the critical need for incorporating novel agents such as bedaquiline into therapeutic strategies against this difficult-to-treat pathogen.